The cell total protein was isolated using RIPA (Applygen Technologies Inc., Beijing, China). Protease inhibitor (CWBIO, Shanghai, China) was added into the RIPA at a ratio of 1:100. After adding RIPA to the cell culture plate, we collected the cells and centrifuged (12,000 r/min) the material at 4 °C for 10 min [26 ]. Protein concentrations were measured on a Thermo Scientific Pierce BCA protein assay kit (Thermo Fisher, Massachusetts, USA) with 1/4 volume of 5 × loading buffer added to the supernatant. A total of 20 μL of protein was blotted using 10% SDS-polyacrylamide gel, then transferred to a polyethylene difluoride (PVDF) membrane (CST, Boston, MA, USA).
After blocking with 5% defatted milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies (1:1,000) against StAR, CYP19A1, CYP11A1, Mfn2, NR5A1/SF-1 (Abcam, Cambridge, UK) and against cyclin B, cyclin D, cyclin E, CDK4 (Santa Cruz, TX, USA). The membrane HRP goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG secondary antibodies (BOSTER, Wuhan, China) were diluted 1:3,000 according to the instructions and incubated for 1 h. Detection was performed using chemiluminescence Western blotting substrate (Santa Cruz, CA, USA) in Image Lab analysis software Image Lab™, (Bio-Rad, Berkeley, CA, USA).
After blocking with 5% defatted milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies (1:1,000) against StAR, CYP19A1, CYP11A1, Mfn2, NR5A1/SF-1 (Abcam, Cambridge, UK) and against cyclin B, cyclin D, cyclin E, CDK4 (Santa Cruz, TX, USA). The membrane HRP goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG secondary antibodies (BOSTER, Wuhan, China) were diluted 1:3,000 according to the instructions and incubated for 1 h. Detection was performed using chemiluminescence Western blotting substrate (Santa Cruz, CA, USA) in Image Lab analysis software Image Lab™, (Bio-Rad, Berkeley, CA, USA).